A triple-blind randomized clinical trial of different associations between dexamethasone and non-steroids anti-inflammatories for preemptive action in third molar extractions

The aim of this study is to evaluate the preemptive analgesic effects of dexamethasone (DEX) alone or combined with non-steroidal anti-inflammatory drugs (NSAIDs) in third molar surgeries. The subjects were divided into five groups (n = 20 teeth/group); subjects received only 8 mg of dexamethasone 1 h before the surgical procedure (DEX group), or in combination with etodolac (DEX + ETO), ketorolac (DEX + KET), ibuprofen (DEX + IBU), loxoprofen (DEX + LOX). Paracetamol 750 mg was provided as the number of rescue analgesics (NRA). Salivary PGE2 expression was measured preoperatively and at 48 h. Edema and Maximum mouth opening (MMO) were measured postoperatively at 48 h and 7 days. A visual analog scale (VAS) was performed postoperatively at 6, 12, 24, 48, 72 h, and 7 days. Salivary expression of PGE2 showed a decrease only for the DEX group. Edema and MMO and NRA consumption showed no significant differences among the groups (P > 0.05). The VAS showed a significantly lower pain perception at 6 h after the surgery for the DEX + ETO and DEX + KET groups (P < 0.05). The combination of DEX and NSAIDS should be considered for preemptive acute postsurgical pain management in third molar surgery. In some drug associations such as dexamethasone 8 mg + NSAIDS (ETO and KET) in the pre-operative time, only a few rescue analgesics are necessary.

Patients and subjects. This study involved 64 subjects presented as healthy subjects fit with the inclusion criteria (ASA I-II: According to those classified by the American Society of Anesthesiologists ASA II are patients with mild systemic disease. Example: Patient with no functional limitations and a well-controlled disease (eg, treated hypertension, obesity with a BMI below 35, frequent social drinker or cigarette smoker). For this, the authors performed an adequate pre-operative anamnesis, as well as requesting laboratory tests and even medical evaluations, when necessary, to define whether the patient had no functional limitations due to mild comorbidity.), never-smokers, between the ages of 16 and 35 years, with an indication of removal of the mandibular third molars with at least 2/3 of the root formed (Class I or II; Position A/B from Pell and Gregory). All surgical procedures required bony removal and tooth sectioning in order to extract the impacted third molars.
Patients evaluated might present with the inclusion criteria established by the authors, which were: Besides that, patients which did not present the necessary characteristics to participate in the study were excluded. The criteria were: www.nature.com/scientificreports/ (f) Patients who have intolerance to the other materials that will be used in the research, such as 0.5% alcoholic chlorhexidine solution, 0.12% chlorhexidine gluconate solution and 4% articaine hydrochloride solution with epinephrine 1: 100,000. (g) Female patients which were in the menstrual period or pregnant period and lactation. (The menstrual  period causes progesterone to act directly on the neurotransmitters GABA (gamma-aminobutyric acid), opioids, serotonin, and catecholamine, in addition to leading to an increase in prolactin, altering glucose metabolism, the function of the hypothalamic-pituitary-adrenal axis. Even causes insulin resistance and mild nutritional and electrolyte deficiencies that may lead to pain threshold, irritability, and increased fluid retention, which can bias some research analyses 18  Randomization. Subject randomization was performed by a researcher (L.P.F.), who selected groups using the envelope system. The envelope contained papers on which the five groups' names were written, and a random selection was performed. If the subject presented with two lower third molars, which was compatible with the inclusion criteria, the subject underwent a double draw and could be allocated to two groups. In those cases, another envelope had two papers identifying the side of the mandibular third molar: right or left, determining the sequence of the groups to be operated. The drugs were given to the subjects by the same researcher. All the drugs were manipulated, the capsules were equally the same and were given in a surgical envelope to avoid the recognize of the drug by the patient and create a bias. The surgeon (G.A.C.M.) and the researcher who conducted the analysis (T.O.B.P.) did not know the groups to groups to which the subjects were assigned, characterizing a triple-blind clinical trial. When a subject had bilateral third molars, the choice to initially operate on the right or left side was also determined by the researcher (L.P.F.) using the envelope system. In this situation, the surgeries were conducted unilaterally with a minimum interval of 21 days between the extraction procedures.
Surgical procedures. The surgical procedures were performed by the same surgeon. All subjects received preoperative antibiotic therapy with 2 g of amoxicillin 1 h before the surgical procedure. Intrabuccal antisepsis was performed with vigorous mouth washing for 1 min with an aqueous solution of 0.12% chlorhexidine digluconate, followed by extraoral antisepsis with 0.5% chlorhexidine alcohol solution. The anesthetic technique employed comprised a regional blockade of the inferior alveolar, buccal, and lingual nerves. This was performed using a reflux syringe with a long 27-gauge gingival needle for injection of 2% mepivacaine hydrochloride with epinephrine 1:100,000 (Mepiadre, DFL) at a maximum volume of 4.5 ml, equivalent to 2.5 tubes.
A triangular linear flap was made with a number 15 scalpel blade in the distal region of the lower second molar in association with a buccal releasing incision in the mesial aspect of the second molar. Following this, mucoperiosteal detachment was performed with periosteal relief and a retractor was used to expose the operative field. Osteotomy was performed using a model 702 carbide-tipped drill bit mounted on a high-speed pen, in addition to abundant irrigation with sterile saline NaCl 0.9%. Dental removal was completed with curved and straight extractors of the Seldin type (n° 2, 1R, or 1L), followed by careful inspection for the removal of pericoronal follicles with curettes and Kelly curve tweezers.
The bone margins were trimmed to remove bone spicules using a bone file, with abundant irrigation using sterile saline NaCl 0.9%. Thereafter, sutures were performed with nylon 5.0 (Ethicon, Johnson & Johnson, Brazil). Surgical interventions were performed in the morning and afternoon (between 08 am and 6 pm) in a temperature environment control.
After the surgical procedure, all subjects received 12 tablets of 750 mg paracetamol (not identified to the subject, in white tablet form) and were advised to take 1 tablet in case of pain, with a minimum interval of 6 h between administrations of the analgesic. Subjects were instructed to recording a log of all relevant variables such as VAS score at each timepoint, and when analgesics were taken.
Immediately after the surgical procedures, the subjects were prescribed a cold, liquid, pasty, hyperproteinbased diet for the first 48 h postoperatively, as well as other aspects of general care, such as avoiding physical exertion and sun exposure. Subjects were advised not to use cold compresses during the postoperative period. Any subject who developed postoperative complications, such as bleeding and alveolar osteitis was treated and Primary outcome. For this study, the primary outcome variable was the assessment of pain (visual analog scale VAS). Number of rescue analgesics (NAR), Salivary PGE2 concentration, edema and MMO were secondary outcomes variables. Pain was measured using a visual analog scale (VAS). During the postoperative period, all subjects received a VAS protocol containing a linear chart with a scale from 0 to 10. The subjects were instructed to point out the level of postoperative pain on the chart at 6, 12, 24, 48, and 72 h and 7 days after the surgical procedure. A VAS score of 0 represented the absence of postoperative pain, whereas a score of 10 represented the worst pain the subject had experienced 12,20 . The weighted average corresponding to the values of every subject in each group was obtained.
Another outcome measure for pain involved the number of analgesics consumed by the subject during postoperative period. Subjects were instructed to record the time of first analgesic consumption. The patients were no induced to consumed or not the drugs, only were informed that were usually drugs destined to relieve pain, edema and trismus and might be used only if were extremely necessary for a rescue way. The weighted average corresponding to the values of every subject in each group was obtained 21 .
The salivary collection was performed before subjects took the preoperative drug, prior to the surgical procedure, and 48 h after the surgical procedure. Saliva was collected directly in a salivate tube (Salivette; Genese Produtos Diagnósticos Ltda, São Paulo, SP, Brazil). The subjects were informed not to eat or brush their teeth in the 2 h preceding the collection procedure. The tubes were properly capped and stored at −20 °C to keep the samples stable. The salivates were centrifuged at 1000 g for one minute, resulting in a clean and fluid saliva sample that was used to determine the PGE2 salivary concentration. Salivary PGE2 was measured by competitive enzymelinked immunosorbent assay (ELISA) method using a commercial kit (Diametra DKO020, Milano, Italy).
Edema measurement was performed using a three-point analysis with a tape measure. Measurements were made of the mandibular angle to the lateral canthus, tragus to labial commissure, and tragus to pogonion at the preoperative, 48 h postoperative, and 7 days postoperative periods (Fig. 1) 22 .The values obtained were recorded in the subjects' individual files. The researcher (T.O.B.P.) who performed these measurements was blinded and also determined all the results of the analyses of this study. To test intra-observer variability and increase measurement precision, all measurements were repeated by the same operator 3 times to ensure reliability of the method and consistency of the operator 23 . To assess postoperative edema, an average of the three-point measurements was calculated at 48-h postoperative and 7-day postoperative.
Maximum mouth opening was evaluated using measurements in millimeters made with a digital caliper (Mitutoyo, Sakado, Japan) between the incisal edge of the upper central incisors and the lower central incisors on the right side. The measurements were performed preoperatively, as well as 48 h and 7 days after the surgical procedure 12,20 . Statistical analysis. All quantitative data underwent a statistical analysis and normality statistical testing (Shapiro-Wilk test) for homogeneity or heterogeneity distribution. For VAS, edema, and MMO, a two-way ANOVA and Tukey posttests were applied, and results were considered significant when P < 0.05. For PEG2 concentration, a one-way ANOVA and Tukey posttests were performed, results were considered significant when P < 0.05. Only NRA failed in the homoscedasticity statistical test, and a Kruskal-Wallis test was applied. An intention to treat analysis was performed for all randomized patients who were included in this study.
Ethical approval. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (#818.680) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent. Informed consent was obtained from all individual participants included in the study.

Results
Demographic data. A total of 125 subjects were screened for eligibility. Sixty-one subjects were excluded.
Fifty subjects did not meet the inclusion criteria and 11 subjects declined to participate in the study. Thus, 64 subjects were randomized and divided into five groups. All 64 subjects received the planned intervention, however, at the follow-up visit, four subjects were excluded due to postoperative dry socket alveolitis and the use of drugs/medicaments not related to the study as described in the exclusion criteria (Fig. 2). Demographic data regarding mean age, sex, and number of teeth removed are shown in Table 1.
After the first postoperative day (24 h, 48 h, 72 h, and 7 days), DEX + ETO showed the lowest pain perception values. However, DEX showed similar values (P > 0.05; Tukey test). DEX + KET maintained its previous average, and DEX + IBU and DEX + LOX showed the worst pain perception values during all postoperative periods (Fig. 3).   (Fig. 6). Only the source time of analysis showed significant changes, in which the edema increased in the first 48 h postoperative, and decreased, at 7 days postoperative, similar to preoperative data.

Maximum mouth opening (MMO).
Only for the source of variation "time of analysis" there was a sig-  (Fig. 7).

Discussion
This study aimed to evaluate whether benefits exist with a combination of NSAIDs plus DEX in decreasing postoperative pain, edema, and MMO following third molar surgery. Prior studies are unclear whether the combination of these drugs could provide an advantage in postoperative period, which justifies the clinical use of various drugs preoperatively. However, the combination of some NSAIDS plus DEX seems to improve symptoms in the first postoperative hours of acute pain. The preemptive use of steroids for third molar surgery, mainly DEX, is well established, and provides positive results regarding a comfortable postoperative period for the patients 8,24,25 . A 2008 clinical study by Laureano-Filho  . Chart regarding prostaglandin salivary E2 (PGE2) concentration. Data showed that DEX group was the only which decreased the PGE2 at 48 h postoperative (80%), while other groups showed an increase of PGE2 concentration at 48 h postoperative. *The value indicated significant differences between the DEX and DEX + IBU groups (P < 0.05-Tukey test). ** The value indicated significant differences between the DEX + KET and DEX + IBU groups (P < 0.05-Tukey test).

Scientific Reports
| (2021) 11:24445 | https://doi.org/10.1038/s41598-021-04068-z www.nature.com/scientificreports/ and colleagues 19 compared a preoperative dose of 4 mg and 8 mg of DEX in third molar surgeries and observed that there was a significant difference between the doses regarding postoperative MMO and edema, but not postoperative pain, as was observed in another study 26 . This lack of effect on pain may be explained by the ineffectiveness of DEX in reducing prostaglandins at the site of an injury 27 . Another possible explanation for this phenomenon is that cortisol naturally inhibits pituitary beta-endorphins, which are potent endogenous analgesic present in the peripheral circulation 28 . Some clinical trials have suggested that the combination of steroids and NSAIDS improves preemptive effects on MMO, edema, and pain compared to steroids alone 4,14 . This study found that the combination of DEX plus several NSAIDS (DEX + ETO, DEX + KET, DEX + IBU, and DEX + LOX) showed no significant difference in the edema and MMO parameters. The exception was the combination of DEX + KET, which showed a significantly lower value for 48-h postoperative MMO compared with DEX alone. These results could be explained because, as noted above, the use of preoperative DEX alone is well established and provides positive results regarding the improvement of postoperative edema and MMO 8,24,25 .
Although data were obtained about postoperative pain, the VAS analysis showed that patients who took preoperative DEX + ETO and DEX + KET exhibited significantly lower pain perception during the first 6 postoperative hours compared with all the groups. At 12 h postoperative, the DEX group showed similar values to the DEX + ETO and DEX + KET groups, whereas the DEX + LOX and DEX + IBU groups maintained higher pain values during all periods. After the first day (24 h), subjects who took DEX + KET showed an increase in pain perception compared with the DEX and DEX + ETO groups, which had lower VAS values during the postoperative period. There was no significant difference between these two groups.
NRA did not significantly differ among the groups; however, it seemed to be lower for the DEX + ETO (average of 1.2 postoperative analgesics) group compared to the other groups. In addition, the DEX + IBU group presented  www.nature.com/scientificreports/ the highest consumption of all groups. However, it is important to highlight that even though NRA was higher for DEX + IBU group, subjects who received this drug preoperatively took an average of three analgesics during the seven postoperative days. This value is well below what is usually prescribed in most practices, which also often include three days of acetaminophen with opioids at an interval of 6 h 29,30 . DEX alone was the only preoperative therapy that decreased the salivary expression of PGE2 48 h after the surgical procedure. The other groups showed an increase in salivary PGE2. However, the DEX + ETO and DEX + KET groups showed a discreet increase, whereas the DEX + IBU and DEX + LOX groups showed a significant increase in PGE2. This data disagrees with the VAS, which showed that the DEX, DEX + ETO, and DEX + KET groups presented similar pain perception values during the entire postoperative period. However, PGE2 is not the only pain mediator released during an inflammation response. Other algogenic substances are generated from arachidonic acids, such as bradykinin, leukotrienes, tumoral necrosis factors (TNF-α) and interleukins 31 . Although the PGE2 expression values were higher, VAS evaluated the subjects' pain perception. Thus, even though the drugs had no effect on PGE2 expression, they might have decreased the subjects' pain perception. Laureano-Filho and colleagues 19 showed that 8 mg of DEX 1 h before surgery decreased the expression of PGE2 and thromboxane A2 at the surgical site, but this had a minimal effect on reported pain the day of the surgery. Similar results were found in another trial with methylprednisolone 125 mg 1 .
There is no consensus about the preemptive combination of DEX and an NSAID. Although trials have shown great results for this therapy, this study observed that not all combinations produced good results. DEX + IBU and DEX + LOX presented poor results for all parameters, and DEX alone seems sufficient to relieve postoperative pain. Currently, the medical field seeks to decrease the number of drugs prescribed to patients. Furthermore, NSAIDs have several adverse effects, such as cardiovascular and gastrointestinal events 32 . Preemptive DEX alone performed well for postoperative pain, edema, MMO and, decreased salivary PGE2, but KET and ETO, when combined with DEX in a preemptive way, showed similar results to DEX alone in terms of VAS scores 7 days after the surgical procedure and significantly decreased patients' pain perception at 6 h after surgery, compared to DEX alone, which resulted in the worst acute pain after the procedure. This demonstrates important benefits for patients' postoperative comfort.
Indeed, ETO and KET have proven effective against pain when prescribed after third molar surgeries 12,33,34 . When combined with DEX, a single preoperative dose of KET and/or ETO seems to significantly reduce acute pain during the first few hours, and there is no need to prescribe any postoperative NSAID except a rescue analgesic. This study presented some limitations. It did not account for other allogenic cytokine expressions did not evaluate other NSAIDs, so more trials should be designed to establish a clinical consensus about this field. However, the use of specific NSAIDs combined with 8 mg of DEX after third molar surgeries might provide patients with postoperative benefits and should be considered in the dentistry routine.

Conclusion
It can conclude that the preemptive use of steroids with NSAIDS assessed in this study, the combinations involving KET and ETO, significantly improved patients' postoperative pain during the first few hours.
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